Important Safety & Liability Notice
The protocols on this page are general educational references compiled from publicly available facility documentation. They are not a substitutefor hands-on training or your institution's specific standard operating procedures (SOPs). Always confirm all protocols, settings, and safety procedures with your local facility staff before operating any equipment. Cryogenic liquids (LN₂, liquid ethane) and high-pressure systems pose serious burn, asphyxiation, and explosion hazards. CryoEM Services assumes no liabilityfor injury, equipment damage, or experimental outcomes resulting from the use of these protocols. Refer to the manufacturer's official documentation for complete safety and operating instructions:
1. Sample Requirements
Ideal cryo-EM samples are monodisperse, concentrated, and freshly purified. Size exclusion chromatography (SEC) immediately before grid preparation is strongly recommended.
2. Grid Selection Guide
Grid choice significantly impacts ice quality and particle distribution. Gold grids reduce beam-induced motion but cost more.
| Grid Type | Material | Mesh | Best For | Notes |
|---|---|---|---|---|
| Quantifoil R1.2/1.3 | Cu or Au | 300 | Standard SPA | |
| Quantifoil R2/2 | Cu or Au | 300 | Large particles | |
| UltrAuFoil R1.2/1.3 | Gold | 300 | High resolution | |
| C-Flat 1.2/1.3 | Cu | 400 | General use | |
| Graphene oxide coated | Cu + GO | 300 | Preferred orientation |
3. Cryo-EM Protein Preparation
Buffer Exchange
Dialyze or desalt into cryo-EM compatible buffer (20 mM HEPES or Tris pH 7–8, 150 mM NaCl). Remove glycerol, sucrose, and high-concentration PEG — these interfere with vitrification.
Concentration
Use centrifugal concentrators (Amicon Ultra, 10–100 kDa MWCO) to reach 0.5–5 mg/mL. Higher concentrations for small proteins (<150 kDa), lower for large complexes.
Quality Control by SEC
Run analytical SEC to verify monodispersity — a single symmetric peak is ideal. Aggregates and degradation products will ruin your grid. Collect the peak fraction and use immediately.
Membrane Proteins
Optimize detergent or nanodisc concentration to avoid excess empty micelles on the grid. LMNG, DDM, and GDN are popular detergents. Nanodiscs (MSP1D1, MSP2N2) eliminate detergent entirely.
Fragile Complexes
For complexes that dissociate at the air-water interface, consider crosslinking with glutaraldehyde via GraFix (gradient fixation) or BS3 chemical crosslinking before grid preparation.
4. Vitrification Equipment Comparison
The Vitrobot Mark IV remains the standard, but newer devices offer advantages for specific use cases.
Vitrobot Mark IV
Thermo FisherChameleon
SPT LabtechLeica EM GP2
LeicaVitroJet
CryoSol5. Vitrobot Mark IV
The Vitrobot is the most widely used plunge-freezing device in cryo-EM. It uses automated filter-paper blotting in a humidity- and temperature-controlled chamber. This protocol is based on standard operating procedures from UW-Madison CEMRC, Harvard Cryo-EM Center, and Stanford S2C2.
📋 Official Thermo Fisher Scientific documentation ↗Default Settings
| Parameter | Value | Notes |
|---|---|---|
| Temperature | 4 °C | |
| Humidity | 100% | |
| Blot time | 3–5 s | |
| Blot force | 0 to +2 | |
| Wait time | 0–5 s | |
| Drain time | 0 s | |
| Blot total | 1 | |
| Sample volume | 3 µL |
Fill Humidifier
Add 45–60 mL of distilled or MilliQ water to the humidifier. Draw back ~10 mL of air with the syringe to create a vacuum in the tubing. Attach humidifier to the chamber.
Mount Filter Paper
Attach fresh Whatman #1 filter paper to both blotting pads using the clipping rings. Ensure the concave side faces the pad. Replace paper every 4–6 grids — reusing degraded paper causes uneven blotting.
Set Parameters & Equilibrate
Enter your settings on the touchscreen (temperature, humidity, blot time, blot force). Allow the chamber to equilibrate for 15–30 minutes until humidity stabilizes at 100%.
Prepare Cryogen
Fill the cryo-container with liquid nitrogen. Place the ethane cup in the center and slowly dispense liquid ethane until the cup is ~⅔ full. Wait until ethane begins to solidify (white slush), then stir with the spider tool to reach a liquid/slush equilibrium. Place the anti-contamination ring. Safety: ethane is extremely flammable — work in a well-ventilated area away from ignition sources. Wear cryogenic gloves and a face shield.
Glow-Discharge Grids
Plasma-clean grids using a PELCO easiGlow (30–60 s at 15 mA, 0.39 mbar) immediately before use. Glow-discharged grids become hydrophobic again within 30–60 minutes — work quickly.
Mount Grid & Apply Sample
Press 'Place Grid' to extend the rod. Clamp the grid in Vitrobot tweezers with carbon/film side facing you. Pipette 3 µL onto the grid — do NOT touch the grid surface with the pipette tip. For back-side application (tomography), apply to the opposite side.
Blot & Plunge
Press 'Continue' to initiate the automated blot → plunge sequence. The filter papers compress from both sides, wick away excess liquid to create a thin aqueous film, then the grid plunges into liquid ethane within milliseconds.
Transfer to Grid Box
Immediately transfer the grid to a pre-cooled grid box submerged in LN₂. Keep the grid below the liquid nitrogen surface at all times — even brief exposure to air causes ice contamination. Use pre-chilled tools to prevent condensation.
Shutdown
Exit the software. Empty and dry the humidifier. Remove used filter paper. Close the ethane tank valves tightly and purge lines per your facility's safety protocol. Allow condensation to evaporate before closing the chamber.
💡 Tips & Tricks
- Replace filter paper every 4–6 grids. Old paper leaves fibers and blots unevenly.
- If ice is consistently too thick, try increasing blot time by 0.5 s before changing blot force.
- For membrane protein samples with detergent, increase blot time by 1–2 s — detergent slows wicking.
- Document every parameter for every grid. Small changes compound — what works Tuesday may not work Thursday.
- If the ethane cup starts to freeze solid, briefly warm it with the spider tool. Plunging into solid ethane produces crystalline ice.
- Back-blotting (applying sample to the carbon side, blotting from the back) helps with adherent cells and tomography samples.
- Pre-screen 1–2 grids by negative stain at 10× lower concentration before committing to cryo conditions.
- Glow discharge both sides of the grid only if you want particles in both ice surfaces (double-sided application).
- Keep a heat block nearby (37 °C) for drying tweezers between grids — moisture on tweezers causes ice contamination.
- The Vitrobot blot force scale is counterintuitive: negative values push pads closer (more force, thinner ice). Start at 0 and adjust ±2.
6. Leica EM GP2
The Leica EM GP2 is an automated plunge freezer offering one-sided blotting, sensor-controlled blot pressure, and a wider environmental chamber. Increasingly popular at facilities like UW-Madison CEMRC as an alternative to the Vitrobot, particularly for tomography samples where back-side blotting is critical.
📋 Official Leica Microsystems documentation ↗Default Settings
| Parameter | Value | Notes |
|---|---|---|
| Temperature | 4–10 °C | |
| Humidity | 90–99% | |
| Blot time | 1–4 s | |
| Blot mode | One-sided / sensor | |
| Post-blot delay | 0–2 s | |
| Cryogen temp | −175 to −185 °C | |
| Sample volume | 3–5 µL |
Initialize the Chamber
Power on the EM GP2 and set temperature and humidity via the touchscreen. Fill the water reservoir for the humidifier. Allow 15–20 minutes for the environmental chamber to stabilize. Verify humidity has reached target using the built-in sensor.
Prepare Cryogen Bath
Fill the secondary container with LN₂. Condense liquid ethane (or ethane/propane mixture) into the primary cryogen cup. The GP2 can use ethane/propane mixtures (37:63 ratio) which remain liquid at LN₂ temperature — eliminating the need to monitor ethane freezing.
Mount Filter Paper
Attach fresh filter paper to the blotting arm using the magnetic ring. For one-sided blotting, paper is mounted on one side only. Verify alignment using the built-in binoculars — the paper should make gentle, even contact with the grid.
Calibrate Blot Position
In sensor mode: the instrument auto-detects when paper contacts the grid. In manual mode: use the 'Adjust' arrows to position the filter paper until it makes light contact with a test grid. This calibration step is critical for reproducibility.
Glow-Discharge Grids
Plasma-clean grids (PELCO easiGlow, 30–60 s, 15 mA). For one-sided blotting, only the sample-application side needs to be hydrophilic.
Load Grid & Apply Sample
Mount grid in tweezers and insert into the chamber clip. Apply 3–5 µL of sample through the side port, then close the port immediately to maintain humidity. The side-port design minimizes sample evaporation compared to top-loading designs.
Blot & Plunge
Initiate the blotting sequence. The GP2 blots from one side, which is critical for cellular tomography — cells on the grid surface are not disturbed. After blotting, the grid automatically plunges into the cryogen bath.
Transfer to Storage
Retrieve the frozen grid from the cryogen bath and transfer to a pre-cooled grid box under LN₂. The GP2 has an integrated transfer station that keeps grids submerged during handling.
💡 Tips & Tricks
- The ethane/propane mixture (37:63) stays liquid at LN₂ temperature — no need to babysit the cryogen like with pure ethane on the Vitrobot.
- One-sided blotting is the GP2's killer feature for tomography — cells on the grid surface are untouched.
- Sensor-controlled blotting gives more reproducible ice thickness than manual force settings.
- The side-port sample application keeps the chamber sealed, reducing evaporation-driven concentration changes.
- For SPA samples, some users find the GP2 produces slightly thicker ice than the Vitrobot — reduce blot time by 0.5 s as a starting point.
- The binocular viewport lets you visually confirm filter paper contact — use it during calibration.
- After your session: remove the cryogen cup, drain remaining LN₂, empty the humidifier, and remove used filter paper. Leave the chamber open to dry.
- If using manual mode, recalibrate the blot position whenever you change grid types — different grid thicknesses affect contact distance.
7. High-Pressure Freezer (Leica EM ICE / HPM100)
High-pressure freezing (HPF) vitrifies specimens up to 200 µm thick by applying ~2,100 bar of pressure simultaneously with rapid cooling. Essential for cells, tissues, and organisms that are too thick for plunge freezing. This protocol covers the Leica EM ICE (successor to HPM100) and EM PACT2 systems used at facilities for cryo-ET and correlative workflows.
📋 Official Leica Microsystems documentation ↗Default Settings
| Parameter | Value | Notes |
|---|---|---|
| Pressure | ~2,100 bar | |
| Cooling rate | >20,000 °C/s | |
| Max specimen thickness | 200 µm | |
| Carrier types | 3 mm / 6 mm Al | |
| Cryoprotectant | 20% BSA or dextran | |
| LN₂ consumption | ~0.5 L/shot |
Prepare Specimen Carriers
Select the appropriate aluminum planchettes (flat/flat or flat/cavity in 3 mm or 6 mm diameter). Clean carriers by sonication in ethanol, rinse with acetone, and air-dry. Coat the inner cavity with 1-hexadecene release agent to facilitate separation after freezing. For cell monolayers, grow cells on carbon-coated sapphire discs (coat with fibronectin, poly-L-lysine, or Matrigel for adhesion). Prepare sapphire discs 24–48 hours before the HPF session.
Load Specimen
For cell pellets: resuspend cells in cryoprotectant (20% BSA in culture medium or 10% dextran in PBS). Load into the cavity of the planchette using a micropipette. For tissue: cut 100–200 µm slices with a vibratome in ice-cold buffer. Place in carrier cavity filled with cryoprotectant. Critical: eliminate ALL air bubbles — any air space causes catastrophic freezing failure. Use a fine needle or hair tool to remove trapped bubbles.
Assemble Carrier Sandwich
Place the second carrier (flat side down) on top to create a sealed sandwich. For the Leica EM ICE, load the assembled carrier into the appropriate specimen pod/cartridge. Ensure the carrier halves are properly aligned.
Initialize the HPF System
Fill the LN₂ dewar to the fill line. Check hydraulic fluid levels (EM ICE). Run a test shot without a specimen to verify the system is primed and the pressure/cooling sequence is functioning. The display should read ~2,100 bar with rapid pressure rise.
Freeze
Insert the loaded cartridge into the freezing chamber. Press the freeze button. The instrument simultaneously pressurizes to ~2,100 bar and jets LN₂ onto the carrier — the entire freeze event takes <30 milliseconds. The carrier is automatically ejected into a LN₂ bath.
Retrieve & Store
Transfer the frozen carrier from the LN₂ bath to cryo-storage using pre-cooled tools. Work quickly — devitrification occurs above −137 °C. Store in LN₂ storage canes or cryo-EM pucks.
Downstream Processing
For cryo-ET: proceed to cryo-FIB milling (Aquilos 2) to create ~150–200 nm lamellae, then image by cryo-TEM. For room-temperature EM: transfer to freeze-substitution (Leica EM AFS2) where ice is replaced by fixative/resin at −90 °C over 24–72 hours.
💡 Tips & Tricks
- Air bubbles are the #1 cause of HPF failure. Fill every void with cryoprotectant — use a dissecting microscope to inspect the loaded carrier before closing.
- For live-cell experiments, minimize time between removing cells from the incubator and freezing — seconds matter for preserving physiological state.
- 20% BSA in growth medium is the most common cryoprotectant — it doesn't permeate cells or alter morphology. Alternative: 10% dextran (40 kDa) in PBS.
- Run 1–2 test shots at the start of each session to ensure consistent pressure curves.
- For correlative light-electron microscopy (CLEM), use the EM PACT2 with the Rapid Transfer System — it transfers from fluorescence microscope to freeze in <5 seconds.
- Sapphire discs must be carbon-coated for cell adhesion AND for easy separation of the carrier after freezing.
- Apply 1-hexadecene release agent to planchette surfaces before loading — it prevents carriers from fusing during freezing and enables clean separation.
- Check pressure logs after each shot — inconsistent pressure curves (below 2,000 bar or slow rise) indicate air contamination, worn seals, or insufficient LN₂.
- Safety: always wear cryogenic gloves and face protection. LN₂ can splash during the freeze cycle and carrier ejection.
8. Single Particle Sample Prep Tips
9. Negative Stain QC Before Cryo
Always screen your sample by negative stain EM before committing to cryo-EM. A 30-minute negative stain session can save days of wasted Krios time on a bad sample.
What to Check
- Monodispersity (no aggregates)
- Expected particle size & shape
- Approximate concentration
- Preferred orientation tendency
Quick Protocol
- Glow-discharge carbon grid
- Apply 3 µL at 10–100 µg/mL
- Stain with 2% uranyl acetate
- Image at 80–120 kV TEM
Red Flags
- Aggregation / clumping
- Degradation products
- Strong preferred orientation
- Concentration too low/high
10. Buffer Compatibility Quick Reference
Not all buffer components are compatible with cryo-EM vitrification. Use this table to check your buffer before grid preparation.
| Component | Verdict | Notes |
|---|---|---|
| HEPES (20 mM, pH 7.5) | ✅ Ideal | Standard cryo-EM buffer |
| Tris (20 mM, pH 7–8) | ✅ Good | pH-sensitive to temperature |
| NaCl (150 mM) | ✅ Good | Standard ionic strength |
| NaCl (>500 mM) | ⚠️ Caution | Can affect ice quality |
| Glycerol (<5%) | ⚠️ Caution | Reduces contrast at higher % |
| Glycerol (>5%) | ❌ Avoid | Prevents vitrification |
| Sucrose (>5%) | ❌ Avoid | Thick ice, low contrast |
| PEG (>1%) | ❌ Avoid | Causes ice artifacts |
| DMSO (>1%) | ❌ Avoid | Prevents proper freezing |
| DDM (0.01%) | ✅ Useful | Helps preferred orientation |
| TCEP/DTT (1–5 mM) | ✅ Good | Reducing agents are fine |
| EDTA (1 mM) | ✅ Good | Chelators are fine |
| Phosphate buffer | ⚠️ Caution | Can precipitate with divalents |
| Imidazole (>50 mM) | ⚠️ Caution | Dialyze out after Ni purification |
11. Cryo-ET / Tomography Sample Preparation
Cryo-electron tomography (cryo-ET) has different sample prep requirements than single particle analysis. Cellular samples, lamellae preparation, and tilt series considerations all factor in.
Cell Culture on Grids
Grow cells directly on gold EM grids (Quantifoil R2/2 or R3.5/1 on Au 200 mesh). Coat grids with fibronectin, poly-L-lysine, or laminin for adhesion. Seed cells 12–24 hours before vitrification to allow spreading. Target thin cell periphery — the region must be <500 nm for electron penetration.
FIB Milling (Cryo-FIB/SEM)
For thicker cells or tissue, use focused ion beam milling at cryo temperatures (Aquilos 2, Arctis) to create ~150–200 nm thin lamellae. This is essential for cellular tomography of interior structures. Lamellae are extremely fragile — handle with care during transfer.
Vitrification for Tomography
Use back-side blotting to preserve cells on the grid surface. Blot from the opposite side to the cells. Gold grids are strongly recommended for tomography — they reduce beam-induced motion during the tilt series. Consider adding gold fiducials (5–10 nm colloidal gold) for tilt series alignment.
Tilt Series Considerations
Ice thickness is critical — thinner is better for tilt series because the effective path length increases as cos(tilt angle). At 60° tilt, the path through 200 nm of ice becomes 400 nm. Target 100–200 nm ice for optimal tilt range.
12. Grid Storage & Shipping
Storage Best Practices
- Store grids in grid boxes under liquid nitrogen at all times
- Label boxes clearly — grid type, sample, date, conditions
- Grids remain viable for months to years if kept below −150°C
- Use dedicated LN₂ storage dewars (not transport dewars)
- Keep a detailed grid log with freezing parameters
Shipping Frozen Grids
- Use a dry shipper (e.g., Taylor-Wharton CX100, MVE SC 4/2V)
- Charge dry shipper with LN₂ 24–48 hours before shipping
- Drain excess LN₂ before air transport (IATA regulation)
- Ship Monday–Wednesday to avoid weekend delays
- Include temperature indicator and tracking number
- Coordinate with receiving facility for immediate transfer
13. Troubleshooting Common Problems
Ice too thick
- Reduce blot time by 0.5–1 s
- Increase blot force by 1 step
- Check filter paper freshness
Preferred orientation
- Add 0.01% DDM detergent
- Use graphene-oxide grids
- Collect with 30° stage tilt
Empty holes / no particles
- Increase protein concentration 2–3×
- Try different grid type
- Reduce blot time (less wicking)
Aggregation on grid
- Use fresh SEC fraction
- Add 0.005% fluorinated detergent
- Check for air-water interface denaturation
Crystalline ice
- Ensure ethane is fully liquid
- Increase plunge speed
- Pre-cool tweezers adequately
Uneven ice gradient
- Replace filter paper (Whatman #1)
- Verify symmetric blotting pads
- Check humidity is at 100%
Frequently Asked Questions
How much protein do I need for cryo-EM?
For a typical single-particle project, you need 3–5 µL at 0.5–5 mg/mL for initial screening (2–3 grids), plus 20–50 µL for optimization and final datasets. Total: approximately 50–200 µg of purified protein.
What buffer should I use?
Common cryo-EM buffers: 20 mM HEPES or Tris pH 7–8, 150 mM NaCl, optional 1–5 mM reducing agent. Avoid: glycerol (>5%), sucrose, high PEG, high detergent concentrations.
Can I ship frozen cryo-EM grids?
Yes. Grids stored in grid boxes under liquid nitrogen can be shipped in dry shippers (e.g., Taylor-Wharton CX100). Grids remain viable for months to years if kept below −150°C.
What is the difference between Vitrobot and Chameleon?
The Vitrobot Mark IV uses filter paper blotting and is the industry standard. The Chameleon (SPT Labtech) uses piezo dispensing onto self-wicking grids, requiring less sample (~50 nL vs 3 µL) and enabling faster freezing.
How do I fix preferred orientation?
Try adding 0.01% DDM or fluorinated Fos-Choline detergent, using graphene-oxide coated grids, or collecting data with stage tilts (20–40°). Multiple strategies can be combined.
Related Resources
Need Help Finding a Facility?
Browse our directory or get a fast-track quote from a private CRO with sample prep services.